Recombinant viruses are generated from the genome of viruses through genetic engineering. This genetic engineering often involves insertion of heterologous DNA including, but not limited to, DNA encoding a therapeutic product into the adenovirus genome. It is to be understood, however, that the term “recombinant virus” is also meant to include virus from which parts of the virus genome have been removed without insertion of heterologous DNA. Another example of a recombinant virus is a chimeric virus containing parts of the genomes of different viruses or of different types of the same virus, such as, e.g., different serotype viruses or viruses with different host animal species specificities.
As used herein, two types of recombinant viruses are discriminated, i.e., replication-deficient virus and replication-competent virus.
In a first type of replication-competent adenovirus, no parts of the adenovirus genome have been removed or parts of the adenovirus genome that are removed do not include parts that are essential for at least one step of the adenovirus infectious life cycle. This type of recombinant adenovirus is, therefore, also defined as a genuine replication-competent adenovirus that has a capacity to replicate in cells like the parental unmodified adenovirus does. In general, the replication of adenoviruses is restricted to cells of a particular animal species or group of animal species. For example, recombinant adenoviruses derived from human adenoviruses can only transverse a complete life cycle in human cells, with very inefficient replication occurring at a high dose in cells of some other species.
A second type of replication-competent adenovirus is the so-called conditionally replicating adenovirus (CRAd). In CRAds, one or more parts of the adenovirus genome are removed, including parts that are essential for at least one step of the adenovirus infectious life cycle under certain physiological conditions (herein also referred to as “first conditions”) but not under certain other physiological conditions (herein also referred to as “second conditions”). The first and second conditions could, e.g., be dictated by the physiological conditions that exist in a particular type of cells (herein also referred to as “first cells”), but not in another type of cells (herein also referred to as “second cells”). Such a first type of cell is, for instance, a cell derived from a particular type of tissue, where the cell contains a protein that is not or much less present in cells from other tissues (second type of cells). An example of a second type of cell is a cell that has lost proper cell growth control, such as, for example, a cancer cell, where the cell either lacks a protein that is present in cells that have not lost proper cell growth control or where the cell has gained expression (or over-expression) of a protein that is not or much less present in cells that have not lost proper cell growth control. Another example of the second conditions is the conditions that exist in a particular stage of the cell cycle or in a particular developmental stage of the cell, where a certain protein is expressed specifically. Thus, CRAds can be designed such that replication thereof is enabled in particular cells, such as cancer cells or a particular type of cancer cells, whereas in normal cells, replication of CRAds is not possible. This is known in the art and reviewed, e.g., by Heise and Kim, J. Clin. Invest. 105(2000):847-851; Alemany et al., Nat. Biotech. 18(2000):723-727; Gomez-Navarro and Curiel, Lancet Oncol. 1(2000):148-158).
In a third type of replication-competent adenovirus, parts of the genome are removed that include the function to be replicated in the target cell, but the function is complemented by inserting one or more functional expression cassettes for heterologous proteins that provide the function in the recombinant adenovirus genome. This type of recombinant adenovirus is referred to herein as a heterologously trans-complemented adenovirus and, therefore, is to be regarded as replication-competent according to the definition presented herein.
The adenovirus replication process constitutes the following steps: (1) infection of the host cell by binding of the adenovirus particle to the cell surface, internalization and transport towards the cell nucleus, and import of the adenovirus DNA genome into the cell nucleus, (2) expression of adenovirus proteins encoded by the early regions in the adenovirus genome, (3) replication of the adenovirus genome, which marks the transition of the early replication phase to the late replication phase, (4) expression of adenovirus proteins encoded by the late regions in the adenovirus genome, (5) assembly of progeny adenovirus particles and inclusion of progeny adenovirus genomes into these particles, and (6) induction of cell death, leading to release of adenovirus progeny from the cell.
During their life cycle, adenoviruses modulate cell-death pathways. In different cell lines, p53-dependent, as well as p53-independent, apoptosis has been documented after adenovirus infection (Teodoro and Branton, J. Virol. 71(1997):1739-1746, and references therein). During the early replication phase, cell death is suppressed to prevent premature cell death, thereby allowing the adenovirus to complete its life cycle in the cell. In contrast, at late stages of infection, cell death and lysis are promoted to release the virus progeny from the cell.
The production of recombinant adenoviruses usually starts with genetic engineering of at least a part of the adenovirus genome by standard molecular biology techniques known in the art. Next, the adenoviral genome, comprised of one or more (in this case overlapping) constructs, is introduced into cells that allow replication of the recombinant adenovirus by DNA transfer methods known in the art. After the recombinant adenovirus has started to replicate in cells into which the recombinant adenovirus genome has been introduced, the recombinant adenovirus can spread to other cells in the culture.
The recombinant adenovirus can also be isolated from the culture medium or from lysates of the cells in which the recombinant adenovirus is replicating. The isolated recombinant adenovirus can then be used to re-infect new cells to further propagate and expand the recombinant adenovirus. In addition, the recombinant adenovirus can be administered to an animal or human body to infect cells in vivo. This administration can be done via several routes including, but not limited to, direct injection into a tissue, oral administration, injection into the blood circulation, inhalation, injection into a body cavity, and application to the surface of a certain body area. Following infection of the cells in vivo, the recombinant adenovirus can replicate and spread to other cells in vivo, provided that the infected cells support replication of the recombinant adenovirus.
Replication-competent adenoviruses will replicate in many different cells in an animal body, provided that they are derived from adenoviruses with the correct species tropism and that the cells express surface receptors for the adenoviruses. Specific cell surface recognition by recombinant adenoviruses, including replication-competent adenoviruses, can be changed. by pseudotyping or targeting (Krasnykh et al., Mol. Ther. 1(2000):391-405; Havenga et al., J. Virol. 76(2002):4612-4620; van Beusechem et al., Gene Ther. 10(2003):1982-1991). CRAds will only replicate in cells in which the particular conditions exist that are required for replication of the CRAd. CRAds are designed to meet the specific requirements for replication in a chosen (first) type of cell and not in other (second) types of cells. This property makes CRAds particularly useful for several embodiments of the present invention where the intent is to treat a disease by specific lytic replication of the recombinant adenovirus, described herein, in diseased cells in an animal or human body, resulting in specific removal of the diseased cells from the body.
Replication-competent viruses, in particular, adenoviruses, are finding increasing utility for the treatment of cancer and other diseases involving inappropriate cell survival. In particular, CRAds have been developed to selectively replicate in and kill cancer cells. Such cancer-specific CRAds represent a novel and very promising class of anticancer agents (reviewed by Heise and Kirn, supra; Alemany et al., supra; Gomez-Navarro and Curiel, supra). The tumor-selective replication of this type of CRAds is achieved through either of two alternative strategies.
In the first strategy, the expression of an essential early adenovirus gene is controlled by a tumor-specific promoter (e.g., Rodriguez et al., Cancer Res. 57(1997):2559-2563; Hallenbeck et al., Hum. Gene Ther. 10(1999):1721-1733; Tsukuda et al., Cancer Res. 62(2002):3438-3447; Huang et al., Gene Ther. 10(2003):1241-1247; Cuevas et al., Cancer Res. 63(2003):6877-6884).
The second strategy involves the introduction of mutations in viral genes to abrogate the interaction of the encoded RNA or protein products with cellular proteins necessary to complete the viral life cycle in normal cells, but not in tumor cells (e.g., Bischoff et al., Science 274(1996):373-376; Fueyo et al., Oncogene 19(2000):2-12; Heise et al., Clin. Cancer Res. 6(2000):4908-4914; Shen et al., J. Virol. 75(2001:4297-4307; Cascallo et al., Cancer Res. 63(2003):5544-5550).
During their replication in tumor cells, CRAds destroy cancer cells by inducing lysis, a process that is further referred to as “oncolysis.” The release of viral progeny from lysed cancer cells offers the potential to amplify CRAds in situ and to achieve lateral spread to neighboring cells in a solid tumor, thus expanding the oncolytic effect. The restriction of CRAd replication to cancer or hyperproliferative cells dictates the safety of the agent, by preventing lysis of normal tissue cells. Currently, CRAd-based cancer treatments are already being evaluated in clinical trials (e.g., Nemunaitis et al., Cancer Res. 60(2000):6359-6366; Khuri et al., Nature Med. 6(2000):879-885; Habib et al., Hum. Gene Ther. 12(2001):219-226).
However, despite very encouraging results from in vitro and animal studies, the anti-cancer efficacy of CRAds as a single agent in humans has been limited (Kim et al., Nature Med. 4(1998):1341-1342; Ganly et al., Clin. Cancer Res. 6(2000):798-806; Nemunaitis et al., Cancer Res. 60(2000):6359-6366; Mulvihill et al., Gene Therapy 8(2001):308-315). Thus, there is a clear need in the field of cancer treatment to increase the potency of replication-competent adenoviruses as oncolytic agents. This could be achieved by enhancing their replication and lysis capacities.
Several approaches aimed at improving the replication and lysis capacities of replication-competent adenoviruses, or at preventing loss of these functions from the wild-type adenovirus, have been taken. It has been shown that it is better to retain the adenovirus E3 region in a replication-competent adenovirus (Yu et al., Cancer Res. 60(2000):4200-4203) or, in case most of the E3 region is deleted, to at least retain the gene encoding the E3-11.6 kDa protein (Tollefson et al., J. Virol. 70(1996):2296-2306; Doronin et al., J. Virol. 74(2000):6147-6155). In addition, replication and cell lysis of replication-competent adenoviruses have been improved by incorporation of cytotoxic genes (Zhang et al., Proc. Natl. Acad. Sci. USA 93(1996):4513-4518; Freytag et al., Hum. Gene Ther. 9(1998):1323-1333; Wildner et al., Gene Ther. 6(1999):57-62). It was also shown that replication-competent adenoviruses are more potent in killing cancer cells when they are deleted of the gene encoding the anti-apoptotic E1B-19 kDa protein (Martin Duque et al., Cancer Gene Ther. 6(1999):554-563; Sauthoff et al., Hum. Gene Ther. 11(2000):379-388).
Recently, we found that oncolysis and release of adenovirus progeny from infected cancer cells can be accelerated by restoring p53 functions in the cancer cells (van Beusechem et al., Cancer Res. 62(2002):6165-6171; PCT International Patent Application WO 03/057892, incorporated by reference herein). Restoration of p53 functions is done by expressing in the cancer cells a restoring factor, i.e., a functional factor of the p53-dependent apoptosis pathway, the function whereof is not or insufficiently expressed in the cancer cells, wherein the restoring factor preferably comprises a protein (WO 03/057892). Hence, the restoring factor is an essential positive component of the p53-dependent apoptosis pathway.
Cancer cells and cell lines are the result of neoplastic transformation. The genetic events underlying neoplastic transformation include activation of proto-oncogenes and inactivation of tumor-suppressor genes. A major player in this respect is the gene encoding the tumor-suppressor protein p53.
The p53 protein is the central coordinator of damage-induced cell-cycle checkpoint control. In a perturbed cell, p53 can induce growth arrest and cell death. p53 exerts these effects by functioning as a specific transcription factor that controls the expression of a large panel of genes involved in growth control, DNA repair, cell-cycle arrest, apoptosis promotion, redox regulation, nitric oxide production, and protein degradation (Polyak et al., Nature 389(1997):237-238; E1-Deiry, Sem. Cancer. Biol. 8(1998):345-357; Yu et al., Proc. Natl. Acad. Sci. USA 96(1999):14517-14522; Hupp et al., Biochem. J. 352(2000):1-17; and references therein).
The induction of cell death by p53 is mediated, at least in part, by activation of pro-apoptotic death genes of the bcl-2 family, such as bax, bak, bad, bid, bik, bim, bok, blk, hrk, puma, noxa and bcl-xs (Miyashita and Reed, Cell 80(1995):293-299; Han et al., Genes Dev. 10(1996):461-477; Zoernig et al., Biochim. Biophys. Acta 1551(2001):F1-F37). On the other hand, anti-apoptotic members of the bcl-2 family, such as bcl-2 itself and bcl-xL, bcl-w, bfl-1, brag-1 and mcl-1 inhibit p53-dependent cell death (Zoernig et al., supra). The anti-apoptotic protein Bax Inhibitor-1 (BI-1) suppresses apoptosis through interacting with bcl-2 and bcl-xL, (Xu and Reed, Mol. Cell 1 (1998):337-346).
The immediate effector proteins of p53, as well as p53 itself, target mitochondria, thereby releasing cytochrome c into the cytosol to activate the caspase cascade via the initiator caspase-9/Apaf-1 complex (Juergensmeier et al., Proc. Natl. Acad. Sci. USA 95(1998):4997-5002; Fearnhead et al., Proc. Natl. Acad. Sci. USA 95(1998):13664-13669; Soengas et al., Science 284(1999):156-159; Marchenko et al., J. Biol. Chem. 275(2000):16202-16212). Negative regulators of the caspase cascade include, but are not limited to, members of the Inhibitor of Apoptosis Protein (IAP) family of proteins, such as cIAP1, cIAP2, cIAP3, XIAP and survivin (Zoemig et al., supra).
The loss of normal function of p53 is associated with resistance to programmed cell death, cell transformation in vitro and development of neoplasms in vivo. In approximately 50% of human cancers, the gene encoding p53 is non-functional through deletion or mutation (Levine et al., Nature 351(1991):453-456; Hollstein et al., Science 253(1991):49-53; Chang et al., J. Clin. Oncol. 13(1995):1009-1022). In many of the other, 50% cancer cells that do express wild-type p53 protein, p53 function is still hampered by the action of a “p53 antagonist.” A “p53 antagonist” is defined herein as a molecule capable of inhibiting p53 function. For example, loss of the tumor-suppressor protein p14ARF or overexpression of the MDM2 protein can lead to functional inactivation of p53 by binding to the MDM2 protein and subsequent degradation (Landers et al., Oncogene 9(1994):2745-2750; Florenes et al., J. Nat. Cancer Inst. 86(1994):1297-1302; Blaydes et al., Oncogene 14(1997):1859-1868; Stott et al., EMBO J. 17(1998):5001-5014; Schmitt et al., Genes Dev. 19(1999):2670-2677). Other nonlimiting examples of molecules that promote p53 degradation include Pirh2 (Leng et al., Cell 112(2003):779-791), COP1(Dornan et al., Nature 429(2004):86-92) and Bruce (Ren et al., Proc. Natl. Acad. Sci. USA 102(2005):565-570).
Another example is functional inactivation of p53 as a result of the antagonizing binding of human papilloma virus (HPV) E6 protein in cervical carcinomas (Scheffner et al., Cell 63(1990):1129-1136) or of herpesvirus-8 latency-associated nuclear antigen (LANA) in Kaposi's sarcoma (Friborg et al., Nature 402(1999):889-894). Yet another example is functional inactivation of p53 as a result of cytoplasmic retention through binding of p53 to Parc (Nikolaev et al., Cell 112(2003):29-40) or to mot-2/mtbsp70/GRP75/mortalin (Wadhwa et al., Exp. Cell Res. 274(2002):246-253).
Furthermore, some molecules can indirectly reduce the amount of functional p53 in a cell and are, therefore, also considered herein as p53 antagonists, although they are not considered members of the p53 pathway. For example, elevated expression of polo-like kinase-1 (plk-1) decreases p53 stability (Liu and Erikson, Proc. Natl. Acad. Sci. USA 100(2003):5789-5794). In addition, even if the p53 function itself is intact, p53-dependent cell death can be hampered due to overexpression of anti-apoptotic proteins acting on the p53 pathway down-stream from p53, such as the anti-apoptotic bcl-2 and IAP family members and BI-1. Another example is p73DeltaN, which binds to p53-responsive promoters competing with p53, thereby antagonizing p53-dependent cell death (Kartasheva et al., Oncogene 21(2002):4715-4727). For the purpose of the invention, the anti-apoptotic proteins acting on the p53 pathway down-stream from p53 are referred to as “p53 pathway inhibitors.” Thus, in many, if not all, cancers in vivo and cancer-derived or immortalized cell lines in vitro, p53-dependent cell death is hampered as a result of one or more lesions in the p53 pathway.
Loss of p53 function has also been documented in other diseases involving inappropriate cell survival, such as, for example, rheumatoid arthritis (Firestein et al., J. Clin. Invest. 96(1995):1631-1638; Firestein et al., Am. J. Pathol. 149(1996):2143-2151; Firestein et al., Proc. Natl. Acad. Sci. USA 94(1997):10895-10900) and vascular smooth muscle cell hyperplasia (Speir et al., Science 265(1994):391-394; Kovacs et al., Am J. Pathol. 149(1996):1531-1539).
Other molecules involved in regulation of programmed cell death include, but are not limited to, members of the death effector domain protein family (reviewed by Tibbetts et al., Nat. Immunol. 4(2003):404-409). It is to be understood that many anti-apoptotic proteins known to be important in the regulation of programmed cell death or cancer cell maintenance do not act on the p53 pathway. They are, therefore, not considered members of the p53 pathway. For example, inhibition of cyclin E, DNA replication initiation proteins, fatty acid synthase, or PAX2 caused apoptosis in cancer cells (Li et al., Cancer Res. 63(2003):3593-3597; Feng et al., Cancer Res. 63(2003):7356-7364; de Schrijver et al., Cancer Res. 63(2003):3799-3804; Muratovska et al., Oncogene 22(2003):6045-6053). Therefore, in all of these targets, the inhibition whereof leads to apoptosis and that are not members of the p53 pathway, are considered anti-apoptotic proteins for the purpose of the invention. Many genes known to be important in the regulation of programmed cell death or cancer cell maintenance have been shown to be targets for anti-cancer therapy (reviewed by Jansen and Zangemeister-Wittke, Lancet Oncol. 3(2002):672-683). Several methods have been used successfully to selectively suppress these targets, including expression of dominant-negative proteins, introduction of small inhibitor molecules, RNA antisense expression and RNA interference. The present invention makes use of RNA interference.
RNA interference (RNAi) is a conserved cellular surveillance system that recognizes double-stranded RNA (dsRNA) and activates a sequence-specific degradation of RNA species homologous to the dsRNA (Hannon, Nature 418(2002):244-251). In addition, RNAi can cause transcriptional gene silencing by RNA-directed promoter DNA methylation and/or histone methylation (Kawasaki and Taira, Nature 431(2004):211-217; Morris et al., Science 305(2004):1289-1292). Furthermore, in some species, RNAi has been implicated in programmed DNA elimination and meiotic silencing (reviewed by Matzke and Birchler, Nature Rev. Genet. 6(2005):24-35). The observation that dsRNA could elicit a potent and specific gene silencing effect was first made in experiments with Caenorhabditis elegans where dsRNA, a byproduct in the generation of antisense RNA, proved to be more effective than antisense RNA itself (Fire et al., Nature 391(1998):806-81 1). In retrospect, this observation offered an explanation for the phenomenon of post-transcriptional gene silencing frequently encountered in transgenic plants (Baulcombe, Plant Mol. Biol. 32(1996):79-88). After these initial reports, RNAi-related processes have been described in almost all eukaryotic organisms, including protozoa, flies, nematodes, insects, parasites, and mouse and human cell lines (reviewed in: Zamore, Nat. Struct. Biol., 8(2001):746-750; Hannon, Nature 418(2002):244-251; Agrawal et al., Microbiol. Mol. Biol. Rev. 67(2003):657-685). By now, RNA interference is the most widely used method to specifically down-regulate genes for functional studies.
The molecular mechanism of RNAi involves the recognition and cleavage of dsRNA into small interfering RNAs (siRNAs) by the RNase III enzymes Dicer and Drosha (Carmell and Hannon, Nat. Struct. Biol. 11(2004):214-218), the incorporation of the siRNAs in a multiprotein complex called RISC (RNA-induced silencing complex), and the degradation of homologous RNA(Caudy et al., Nature 425(2003):411-414). The siRNAs perform an essential role in guiding RISC to the target mRNA. siRNAs consist of double-stranded 21 to 23 nucleotide RNA duplexes carrying two nucleotide 3′-OH overhangs that determine, in part, the efficacy of gene silencing (Elbashir et al., Genes Dev. 15(2001):188-200).
During the incorporation of the siRNA into the RISC complex, the siRNA is unwound and only one strand is assembled into the active RISC complex. This process is asymmetric in nature and RISC preferentially accepts the strand of the siRNA that presents the less stable 5′ end (Khvorova et al., Cell 115(2003):209-216; Schwarz et al., Cell 115(2003):199-208). This has important ramifications in the selection of siRNA sequences because only siRNAs from which the antisense strand (with regard to the targeted mRNA) is assembled into RISC will be effective. Guidelines have been proposed for the selection of highly effective siRNA sequences based on the free energy profile of the siRNA sequences (Khvorova et al., Cell 115(2003):209-216; Schwarz et al., Cell 115(2003):199-208; Reynolds et al., Nat. Biotechnol. 22(2004):326-330; Ui-Tei et al., Nucleic Acids Res. 32(2004):936-948).
Application of dsRNA to silence expression of genes in mammalian cells lagged behind due to the occurrence of a general response triggered by dsRNA molecules larger than 30 basepairs. This response is mediated by dsRNA-activated protein kinase (PKR) and 2′, 5′ OligoA-synthetase/RNAseL and results in a shut down of translation followed by apoptosis (Kumar and Carmichael, Microbiol. Mol. Biol. Rev. 62(1998):1415-1434; Gil and Esteban, Apoptosis 5(2000):107-114). Therefore, initially, application of RNAi by long dsRNA was confined to mammalian cells that lack the PKR response: i.e., embryonic cells (Billy et al., Proc. Natl. Acad. Sci. USA 98(2001):14428-14433; Svoboda et al., Development 127(2000):4147-4156). In a breakthrough experiment, Elbashir and coworkers showed that chemically synthesized siRNAs, resembling the siRNAs produced by Dicer and Drosha, induced gene-specific silencing in cultured mammalian cells without triggering the PKR response (Elbashir et al., Nature 411(2001):188-200). Synthesized siRNAs are now widely used as a tool to study the function of individual genes offering a convenient and rapid method to silence genes (McManus and Sharp, Nat. Rev. Genet. 3(2002):737-747). However, the transient nature of the silencing effect and the difficulty of delivering synthetic siRNAs in vivo restrict the utility of this approach.
A further means of generating siRNAs is to express small RNA molecules inside the cell, either by co-expression of sense and antisense RNAs (Zheng et al., Proc. Natl. Acad. Sci. USA 101(2004):135-140; Miyagishi et al., Nat. Biotechnol. 20(2002):497-500; Lee et al., Nat. Biotechnol. 20(2002):500-505), or as a single transcript that forms a stem-loop structure. The latter RNA molecules are generally referred to as short hairpin RNAs (shRNAs) and typically consist of a 19 to 29 nucleotide stem containing complementary sense and antisense strands and a loop of varying size. shRNAs generated inside the cell are processed by Dicer to form siRNAs and are capable of inducing RNAi.
A variety of promoters have been used to drive the expression of shRNAs. RNA polymerase III promoters are especially suited because they have well-defined initiation sites and a termination site consisting of a stretch of at least four consecutive thymidine nucleotides. The RNA polymerase III (polIII) promoters H1, U6 and tRNA(Val) have been successfully used to express shRNAs (Brummelkamp et al., Science 296(2002):550-553; Paddison et al., Genes Dev. 16(2002)948-958; Kawasaki and Taira, Nucleic Acids Res. 31 (2003):700-707).
Recently, polIII-based drug-inducible shRNA expression cassettes have been developed that permit the conditional suppression of genes in mammalian cells (Wiznerowicz and Trono, J. Virol. 77(2003):8957-8961; Gupta et al., Proc. Natl. Acad. Sci. USA 101(2004):1927-1932). RNA polymerase II promoters (polII) are less suited to express functional shRNAs and initial attempts failed to induce silencing (Paddison et al., Genes Dev. 16(2002)948-958). However, successful use of the polII CMV promoter was reported by using a minimal CMV promoter and a modified polyA signal (Xia et al., Nat. Biotechnol. 20(2002):1006-1010) or by using ribozyme-mediated cleavage of the transcript (Kato and Taira, Oligonucleotides 13(2003):335-343; Shinagawa and Ishii, Genes Dev. 17(2003):1340-1345).
Reports that shRNAs expressed from.plasmids could trigger RNAi allowed the use of viral vectors. Retroviral or lentiviral delivery into mammalian cells leads to stable integration of the shRNA-expression cassette in the genome and long-term, sustained gene suppression and is frequently used (e.g., Brummelkamp et al., Science 296(2002):550-553; An et al., Hum. Gene Ther. 14(2003): 1207-1212).
Adenoviral vectors infect dividing and non-dividing cells but remain episomal. Non-replicating adenoviral vectors expressing shRNAs have been shown to induce silencing of target genes in vitro and in vivo (Xia et al., Nat. Biotechnol. 20(2002):1006-1010; Arts et al., Genome Res. 13(2003):2325-2332; Shen et al., FEBS Lett. 539(2003):111-114; Zhao et al., Gene 316(2003):137-141; WO 2004/013355). So far, RNAi with viral vectors has only been done using replication-deficient viral vectors. For clarity, it is repeated here that the present invention relates only to replication-competent virus. It has not been suggested before to employ RNAi in the context of a replication-competent virus because this is not obvious. RNAi has been recognized as a cellular defense mechanism against viral infection and this had led many viruses to evolve molecules that inhibit RNAi (Cullen, Nature Immunol. 3(200):597-599; Roth et al., Virus Res. 102(2004):97-108). Therefore, prior to the present invention, there was reason to assume that the process of RNAi would be hampered in cells in which a virus is replicating. The results disclosed herein, which could not be predicted from prior literature, demonstrate that RNAi can be successfully employed in the context of a replication-competent virus.
RNA interference in mammalian cells is now widely used to analyze the function of individual genes. Numerous genes have been successfully silenced in mammalian cells, either by transfection of chemically synthesized siRNA or by expression of shRNAs. Classes of genes targeted include genes involved in signal transduction, cell-cycle regulation, development, cell death, etc. (Milhavet et al., Pharmacol. Rev. 55(2003):629-648). Systematic studies for delineating gene function on a genome scale are feasible when large libraries targeting human genes are available. Large libraries of chemically synthesized siRNAs are already commercially available (from Dharmacon and Qiagen), which cause strong, but transient, inhibition of gene expression.
By contrast, vector-expressed shRNAs can suppress gene expression over prolonged periods. Two research groups (Berns et al., Nature 428(2004):431-437; Paddison et al., Nature 428(2004):427-431) independently constructed and reported on an shRNA-based library covering 7,914 and 9,610 human genes, respectively. Both libraries use polIII promoters and a retroviral vector based on self-inactivating murine-stem-cell virus. These libraries will aid in identifying gene function in mammalian cells using high-throughput genetic screens. Berns et al. (supra) already used their library to identify new components of the p53 pathway by screening for inhibition of cell senescence. It is expected that synthetic lethal high-throughput screenings will be performed to evaluate shRNAs for their ability to kill engineered tumorigenic cells but not their isogenic normal cell counterparts (Brummelkamp and Bernards, Nat. Rev. Cancer 3(2003):781-789). Thus, identification of selective anti-cancer shRNAs is foreseen.